Natural Antisense Makes Sense for Gene-specific Activation in Brain

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Correspondence: Matthew JA Wood, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. E-mail [email protected] The upregulation of specific genes in vivo has been an elusive goal for gene therapy when compared with the wide repertoire of methods available to silence genes or modify mRNA splicing patterns. In the latest issue of Nature Biotechnology, Modarresi and colleagues1 accomplished in vivo upregulation of brain-derived neurotrophic factor (BDNF), a relevant therapeutic target for a number of neurodegenerative diseases. Rather than using small molecules or microRNA inhibitors, which could lead to activation of off-target genes, Modarresi et al.1 upregulated BDNF by inhibiting a natural antisense transcript (NAT) in response to the local delivery of oligonucleotides to the central nervous systems of mice. It is now clear that mammalian genomes are much more extensively transcribed than was once thought, and that the vast majority of cellular transcriptional output is noncoding RNA. Katayama et al.2 reported widespread antisense transcription in the human and mouse genomes and showed, for the first time, that targeting antisense transcripts with small interfering RNAs influenced the expression of overlapping sense mRNA transcripts. As such, NATs function to regulate expression of neighbouring genes in cis. Further studies from the laboratories of David Corey, Kevin Morris, and Long-Cheng Li demonstrated that antisense transcripts are epigenetic regulators of their corresponding sense strand protein-coding genes, and in the cases of the progesterone receptor and the tumor suppressor p21, targeting these transcripts with small interfering RNAs resulted in a loss of epigenetic repression and consequently gene activation.3,4 More recently, a landmark in vivo study by Turunen et al.5 reported transcriptional activation of vascular endothelial growth factor by lentiviral expressed promoter-targeting short hairpin RNAs in a mouse hindlimb ischemia model, thus demonstrating the therapeutic relevance of this gene activation approach. However, the present study by Modarresi et al.1 is the first demonstration of oligonucleotide-mediated transcriptional gene activation in vivo and is an elegant and important step towards translating this approach into novel molecular therapies, especially for neurological disorders. Targeting the NAT of BDNF to increase BDNF levels in the central nervous system has therapeutic potential. BDNF plays a central role in neurogenesis, neuronal development, and synaptic plasticity. Neurogenesis occurs mainly during development, but in adulthood it is involved in the consolidation of memory for which BDNF is also a critical factor.6 Higher levels of BDNF have also been found to be predictors of a slower cognitive decline in Alzheimer’s disease patients.7 Additionally, activation of the BDNF signaling pathway is responsible, at least in part, for the neuroprotective effects of physical exercise in Parkinson disease, promoting neuronal survival, and facilitating the recovery of brain functions after injury.8 However, little is known about the function of this NAT in humans and Modarresi et al.1 now identify this transcript in mouse for the first time. This study showed that inhibiting the NAT of BDNF stimulated neuronal outgrowth in vitro and improved neuronal survival and proliferation in vivo as a direct result of BDNF gene activation and increased BDNF protein levels. Additionally, the authors showed in vitro activation for two other genes using the same approach: glial-derived neurotrophic factor and ephrin receptor B2. Modarresi et al.1 tested small interfering RNAs and an array of oligonucleotides composed of locked nucleic acids9 either mixed with 2′OMe RNA chemistry for steric block or designed as gapmers to induce RNase H cleavage of complementary NATs. The locked nucleic acid gapmers proved to be most potent oligonucleotide chemistry. Locked nucleic acid bases increase the affinity of the oligonucleotide for the target, make them more resistant to nucleases and already have a successful record for in vivo application.9 Therefore, the best locked nucleic acid gapmer was selected and subsequently evaluated in vivo by intracerebroventricular delivery using an osmotic minipump. Significant increases in BDNF mRNA and protein levels were detected in both frontal cortex and hippocampus. In contrast to the positive data in these brain regions, Modarresi et al.1 observed that the levels of BDNF NAT and BDNF mRNA were unaltered in the hypothalamus, and explained this by suggesting that oligonucleotide delivery was more effective to regions immediately adjacent to the third ventricle. However, the hypothalamus is in very close proximity to the third ventricle; thus alternative explanations for this lack of response could include differential expression of BDNF or its corresponding NAT, which the authors have shown to have highly tissue-specific patterns of expression. Since regulation of gene expression by NATs can be either transcriptional or post-transcriptional, Modarresi et al.1 Commentary